Bile acids salts which are organic acids derived from cholesterol are natural ionic detergents that play a pivotal role in the absorption, transport, and secretion of lipids. In bile acid chemistry, the steroid nucleus of a bile acid salt has the perhydrocyclopentano phenanthrene nucleus common to all perhydrosteroids. Distinguishing characteristics of bile acids include a saturated 19-carbon sterol nucleus, a beta-oriented hydrogen at position 5, a branched, saturated 5-carbon side chain terminating in a carboxylic acid, and an alpha-oriented hydroxyl group in the 3-position. The only substituent occurring in most natural bile acids is the hydroxyl group. In most mammals the hydroxyl groups are at the 3, 6, 7 or 12 positions.
The common bile acids differ primarily in the number and orientation of hydroxyl groups on the sterol ring. The term, primary bile acid refers to these synthesized de novo by the liver. In humans, the primary bile acids include cholic acid (3α, 7α, 12α-trihydroxy-5β-cholanic acid) (“CA”) and chenodeoxycholic acid (3α, 7α-dihydroxy-5β-cholanic acid) (“CDCA”). Dehydroxylation of these bile acids by intestinal bacteria produces the more hydrophobic secondary bile acids, deoxycholic acid (3α, 12α-dihydroxy-5β-cholanic acid) (“DCA”) and lithocholic acid (3α-hydroxy-5β-cholanic acid) (“LCA”). These four bile acids CA, CDCA, DCA, and LCA, generally constitute greater than 99 percent of the bile salt pool in humans. Secondary bile acids that have been metabolized by the liver are sometimes denoted as tertiary bile acids.
Keto-bile acids are produced secondarily in humans as a consequence of oxidation of bile acid hydroxyl groups, particularly the 7-hydroxyl group, by colonic bacteria. However, keto-bile acids are rapidly reduced by the liver to the corresponding α or β-hydroxy bile acids. For example, the corresponding keto bile acid of a CDCA is 7-keto lithocholic acid and one of its reduction products with the corresponding β-hydroxy bile acid is ursodeoxycholic acid (3α-7β-dihydroxy-5β-cholanic acid) (“UDCA”), a tertiary bile acid.
UDCA, a major component of bear bile, has been used for the treatment of and the protection against many types of liver disease for a little over 70 years as a major pharmaceutical agent. Its medicinal uses include the dissolution of radiolucent gall stones, the treatment of biliary dyspepsias, primarily biliary cirrhosis, primary sclerosing choplangitis, chronic active hepatitis and hepatitis C. In other mammalian species, bile acids containing a 6β-hydroxyl group, which are found in rats and mice, are known as muricholic acid; 6α-hydroxy bile acids produced by swine are termed hyocholic acid and hyodeoxycholic acids. 23-hydroxy bile acids of aquatic mammals are known as phocecholic and phocedeoxycholic acids.
Typically, more than 99 percent of naturally occurring bile salts secreted into human bile are conjugated. Conjugates are bile acids in which a second organic substituent (e.g. glycine, taurine, glucuronate, sulfate or, rarely, other substituents) is attached to the side chain carboxylic acid or to one of the ring hydroxyl groups via an ester, ether, or amide linkage. Therefore, the ionization properties of conjugated bile acids with glycine or taurine are determined by the acidity of the glycine or taurine substituent.
Free, unconjugated, bile acid monomers have pKa values of approximately 5.0. However, pKa values of glycine conjugated bile acids are on average 3.9, and the pKa of taurine conjugate bile acids are less than 1.0. The effect of conjugation, therefore, is to reduce the pKa of a bile acid so that a large fraction is ionized at any given pH. Since the ionized salt form is more water soluble than the protonated acid form, conjugation enhances solubility at a low pH. Free bile acid salts precipitate from aqueous solution at pH 6.5 to 7. In contrast, precipitation of glycine conjugated bile acid occurs only at pH of less than 5. Taurine conjugated bile acids remain in aqueous solution under very strongly acidic conditions (lower than pH 1). However, in the gastric pH range, certain bile acids such as UDCA and CDCA are no longer soluble.
Conjugation of the side chain of a bile acid with glycine or taurine has little influence on the hydrophobic activity of fully ionized bile salts. More hydrophobic bile salts exhibit greater solubilizing capacity for phospholipid and cholesterol and are consequently better detergents. More hydrophobic bile salts are also more injurious to various membranes, both in vivo and in vitro.
Natural bile salt pools invariably contain multiple bile acid salts. Mixtures of two or more bile salts of differing hydrophobic activity may behave as a single bile salt of an intermediate hydrophobic activity. As a result, detergent properties and the toxicity of mixtures of two bile acids of differing hydrophobic activity often are intermediate between the individual components. Biologic functions and biologic properties of bile acids resulting from their amphiphillic properties are as follows:    1. Ursodeoxycholic acid is a useful immuno-modulating agent.    2. Ursodeoxycholic acid inhibits deoxycholic acid-induced apoptosis by modulating mitochondrial transmembrane potential and reactive oxygen species production.    3. Ursodeoxycholic acid inhibits induction of nitric oxide synthase (NOS) in human intestinal epithelial cells and in vivo.    4. The hydrophilic nature of ursodeoxycholic acid confers cytoprotection in necroinflammatory diseases of the liver.    5. Ursodeoxycholic acid significantly improves transaminases and cholestatic enzymatic indices of liver injury in chronic hepatitis.    6. Bile acids substantially inhibit the growth of H. pylori.     7. Ursodeoxycholic acid is the most potent pepsin inhibitor among bile acids.    8. High levels of bile acids remarkably inhibit the proliferation of hepatitis C virus.    9. Ursodeoxycholic acid has cell membrane stabilizing properties.    10. Ursodeoxycholic acid alleviates alcoholic fatty liver.    11. Ursodeoxycholic acid has a vasodilative effect on the systemic vascular bed but altered neither pulmonary vascular function nor cardiac functions.    12. Bile acid synthesis from cholesterol is one of the two principal pathways for the elimination of cholesterol from the body.    13. Bile flow is generated by the flux of bile salts passing through the liver. Bile formation represents an important pathway for solubilization and excretion of organic compounds, such as bilirubin, endogenous metabolites, such as emphipathic derivatives of steroid hormones; and a variety of drugs and other xenobiotics.    14. Secretion of bile salts into bile is coupled with the secretion of two other biliary lipids, phosphatidylcholine (lecithin) and cholesterol. Coupling bile salt output with the lecithin and cholesterol output provides a major pathway for the elimination of hepatic cholesterol.    15. Bile salts, along with lecithin, solubilize cholesterol in bile in the form of mixed micelles and vesicles. Bile salt deficiency, and consequently reduced cholesterol solubility in bile, may play a role in the pathogenesis of cholesterol gallstones.    16. Bile acids are thought to be a factor in the regulation of cholesterol synthesis. At present, it is not certain whether they regulate the cholesterol synthesis by acting directly on the hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase or indirectly by modulating the cholesterol absorption in the intestine.    17. Bile salts in the enterohepatic circulation are thought to regulate the bile acid synthesis by suppressing or derepressing the activity of cholesterol 7-hydroxylase, which is the rate-limiting enzyme in the bile acid biosynthesis pathway.    18. Bile acids may play a role in the regulation of hepatic lipoprotein receptors (apo B.E.) and consequently may modulate the rate of uptake of lipoprotein cholesterol by the liver.    19. In the intestines, bile salts in the form of mixed micelles participate in the intraliminal solubilization, transport, and absorption of cholesterol, fat-soluble vitamins, and other lipids.    20. Bile salts may be involved in the transport of calcium and iron from the intestinal lumen to the brush border.
Recent drug delivery research concerning the characteristics and biofunctions of naturally occurring bile acid as an adjuvant and/or a carrier has focused on the derivatives and analogs of bile acids and bile acids themselves as novel drug delivery systems for delivery to the intestinal tract and the liver. These systems exploit the active transport mechanism to deliver drug molecules to a specific target tissue by oral or cystic administration. Thus, if bile acids or bile acid derivatives are rapidly and efficiently absorbed in the liver and, consequently, undergo enterohepatic cycling, many potential therapeutic applications are foreseen including the following: improvement of the oral absorption of an intrinsically, biologically active, but poorly absorbed hydrophillic and hydrophobic drug; liver site-directed delivery of a drug to bring about high therapeutic concentrations in the diseased liver with the minimization of general toxic reactions elsewhere in the body; and gallbladder-site delivery systems of cholecystographic agents and cholesterol gallstone dissolution accelerators. As an example, in 1985, Drs. Gordon & Moses et al. demonstrated that a therapeutically useful amount of insulin is absorbed by the nasal mucosa of human beings when administered as a nasal spray with common bile salts such as DCA, UDCA, CDCA, CA, TUDCA, and TCDCA. See Moses, Alan C., et al., Diabetes vol. 32 (November 1983) 1040–1047; Gordon, G. S., et al., Proc. Nat'l Acad. Sci. USA, vol. 82 (November 1985) 7419–7423. In their experiment, bile acids produced marked elevations in serum insulin concentration, and about 50 percent decreases in blood glucose concentrations. However, this revolutionary nasal spray solution dosage form with bile acids (salts) as an adjuvant could not be developed further and commercialized, because the nasal spray solution must be prepared immediately prior to use due to the precipitation of bile acid salt and the instability of insulin at pH levels between 7.4 and 7.8. Moreover, as indicated in this disclosure, ursodeoxycholic acid as an adjuvant could not be used because of its insolubility at pH between 7.4 and 7.8.
Bile acid salts and insulin, thus, appear to be chemically and physically incompatible. The pH of commercial insulin injection solutions is between 2.5 and 3.5 for acidified dosage forms and is between 7.00 and 7.4 for neutral dosage forms. Dosage forms of bile acid salts prepared by conventional techniques have been unable to overcome problems with bile precipitation at these pH levels and insulin is unstable at a pH of 7.4 or higher. Therefore, safe and efficient preparations of any solution dosage forms of insulin with bile acid (salt) are not commercially available at this time.
Heparin, a most potent anticoagulant, is widely used in the treatment of and in the prevention of thromboembolism. However, heparin treatment is usually limited to hospitalized patients since this drug is given only by injection. Alternate routes which have been attempted are an intrapulmonary spray, suppositories, and enema. According to numerous publications, for heparin absorption through the gastrointestinal mucosa to be facilitated, the preparations should be in acidic condition. According to Dr. Ziv, Dr. Eldor et al., heparin was absorbed through the rectal mucosa of rodents and primates only when administered in solutions containing sodium cholate or sodium deoxycholate. See Ziy E. et al., Biochemical Pharmacology, vol. 32, No. 5, pp. 773–776 (1983). However, heparin is only stable under acidic conditions. Bile acids are particularly not soluble in acidic conditions. Therefore, due to their incompatible characteristics, commercial dosage forms of bile acids with heparin are not presently available.
Drug delivery systems involving bile acids can provide liver-specific drug targeting which is of major interest for drug development since standard pharmacological approaches to liver diseases have been frustrated by the inadequate delivery of active agents into liver cells as well as non specific toxicity towards other organs. For example, the liver-specific delivery of a drug is necessary for inhibitors of collagen synthesis for the treatment for liver fibrosis in order to avoid unspecific and undesired side-effects in extrahepatic tissues. Furthermore, for the treatment of cancer of the biliary system, high drug levels must be achieved in the liver and the biliary system, whereas in extrahepatic tissues low drug concentrations are desired to minimize the cytoxicity of the cytostatics to normal non-tumor cells. Dr. Kramer, Dr. Wess et al. demonstrate that hybrid molecules formed by covalent linkages of a drug to a modified bile acid molecule are recognized by the Na+-dependent bile acid uptake systems in the liver and the ileum. See U.S. Pat. No. 5,641,767. Even if bile acid salts and their derivatives act as shuttles for specific delivery of a drug to the liver, as already mentioned above, there are enormous risks to the development of the derivatives of bile acids or bile acid salts as carriers because new derivatives of bile acids or bile acid salts formed by covalent linkages of a drug to bile acid must be tested for its pharmacology, toxicity and clinical effectiveness. Thus, the development of preparations in which a drug can be absorbed with bile acids or bile acid salts from the places which contain the excessive bile acids in the intestine is far easier and far more valuable than the development of the new bile acid derivatives because less testing is required.
In spite of the extremely valuable therapeutic activities and the long historic medical uses of bile acids as therapeutically active agents and as carriers and/or adjuvants based on the already mentioned biological properties and functions of bile acids, the commercial administration of bile acids is limited to pharmaceutical formulations with a solid form of bile acid which are in tablet, capsule and suspension. This is due to the insolubility of bile acid in aqueous media at pH from approximately 1 to 8. This is also due to bile's extremely bitter taste and equally bitter after-taste which lasts several hours. Ursodeoxycholic acid, chenodeoxycholic acid, and lithocholic acid are practically insoluble in water. Deoxycholic acid and cholic acid have solubilities of 0.24 g/L, and 0.2 g/L, respectively. Tauroursodeoxycholic acid, taurochenodeoxycholic acid, and taurocholic acid are insoluble in hydrochloric acid solution. The few aqueous dosage forms that are available are unstable, and have very limited uses because of pH control and maintenance problems. Moreover, some commercial pharmaceutical dosage forms of bile acids have been shown to have scant bioavailability as described in European Journal of Clinical Investigation (1985) 15, 171–178. Bile acid, especially ursodeoxycholic acid is poorly soluble in the gastro-duodeno jejunal contents of fasted subjects. From 21% to 50% of the ingested doses were recovered in solid form because of the unpredictable variations in the very slow progressive solubilization of solid ursodeoxycholic acid in the gastrointestinal track. Bile acids, particularly ursodeoxycholic acid, deoxycholic acid, chenodeoxycholic acid, cholic acid, hyodeoxycholic acid, 7-keto lithcholic acid, tauroursodeoxycholic acid, and taurochenodeoxycholic acid among others, are especially insoluble in the gastric juices and in aqueous hydrochloric acid solution. However, the solubility of bile acids increase with the increase of the pH in the intestine very slowly and incompletely, and eventually the bile acids become soluble at pH between 8 and 9.5.
To overcome this slow and inefficient absorption process in the intestine due to the incomplete and slow solubilization of bile acids, many newly developed pharmaceutical formulations have been prepared, such as delayed release dosage forms with water soluble solid bile acids which are often strongly alkaline. These newly developed pharmaceutical dosage forms are enterosoluble-gastroresistant. These enterosoluble-gastroresistant dosage forms remain intact in gastric juices in the stomach, but are dissolved and release the strongly alkaline solid bile salts of the formulations at the targeted area, within a limited time once they reach the small intestine.
These types of dosage forms, of course, showed better bioavailability than presently commercialized dosage forms as described in U.S. Pat. No. 5,380,533. However, it is extremely difficult and very costly to prepare the precise delayed release dosage forms which can release therapeutically active components by disintegration, dissolution and diffusion at the desired area within a limited time. According to U.S. Pat. No. 5,302,398, the absorption test of the gastroresistant enterosoluble dosage forms of bile acids, particularly ursodeoxycholic acid in man show that its absorption increases a value of about 40 percent in comparison with administering the same amount in current commercial dosage forms. Its maximum hematic concentrations are on average three times higher, and are reached faster than with the commercial formulations. Any dosage forms of bile acid formula must be capable of releasing bile acids in a known and consistent manner following administration to the patient. Both the rate and the extent of release are important, and should be reproducible. Ideally, the extent of release should approach 100 percent, while the rate of release should reflect the desired properties of the dosage form.
It is a well-known fact that solution dosage forms of drugs show significantly improved rates and extents of absorption, compared to the same drug formulated as a tablet, capsule, or suspension. This is because solution dosage forms are chemically and physically homogeneous solutions of two or more substances. Moreover, the specially designed solution dosage forms which can maintain the solution systems without breaking down under any pH conditions are ready to be diffused in the desired area for immediate and complete absorption, whereas tablets, capsules or delayed release formulations must invariably undergo disintegration, dissolution and diffusion at the desired area within a limited time. Unpredictable variations in the extent and rate of release of bile acids by the disintegration, dissolution and diffusion of delayed or immediate release dosage forms having pH-dependent instability result in the slow and inefficient bile absorption and reduced bioavailability.
The luminal surface of the stomach is coated with a thick layer of protective mucus. The mucus gel coating maintains a pH gradient from the intraluminal compartment to the apical membrane and is believed to contribute to the phenomenon of cytoprotection. H. pylori infection occurs on the luminal surface of the stomach mucosa within the mucus, on the epithelial surface, and within the gastric pints. Bacterial enzymes are believed to degrade the mucus glycoprotein network and reduce the polymers to monomers (or subunits) such that the mucus can no longer exist as a gel. In addition, mucinogenesis is reduced and the mucosa becomes susceptible to the erosive effects of acid. This condition may lead to gastritis and peptic ulcers.
Bismuth compounds have gained increasing interest in the therapeutic treatment of gastro-duodenal disorders and especially in the eradication of Helicobacter pylori, a bacterium thought to be involved in the etiology of the disease. Many oral preparations of bismuth have been used. The various preparations appear to differ in clinical efficacy as well as pharmacokinetics. The inorganic salts used have included subnitrate, subcarbonate, subgallate, tartarate, citrate and subsalicylate. The commercial preparations have generally been available over the counter and have often contained other compounds in addition to the bismuth salt. The commercial preparations have been used successfully in the treatment of both gastric and duodenal ulcer disease. These preparations have proved as effective as the histamine H2 antagonists in the treatment of gastric and duodenal ulcers and have been associated with lower relapse rates after cessation of therapy. The lower relapse rate after initial healing with bismuth preparations have been attributed to its ability to eradicate H. pylori and to moderate the gastroduodenitis associated with infection by this organism. Long-term eradication of H. pylori is more likely when bismuth preparation is administered along with antibiotics or antiseptics (local delivery) such as bile acids.
A variety of antibiotics and antiseptics display good activity against H. pylori in vitro. Yet when tested as single agents in clinical studies, they do not succeed in eradicating the organism. Failure of therapy and relapse are very common. The reason for this discrepancy between in vitro and clinical results has not been established. Possible explanations are poor penetration of the compounds into gastric mucus, destruction at acid pH, insolubility in acidic environment, and combinations thereof. Consequently, administration of high doses of antimicrobial agents on a daily basis is necessary for H. pylori eradication. The efficacy of this course of therapy is hindered by poor patient compliance due to adverse effects such as diarrhea, nausea, retching and breakdown of normal intestinal flora.
Another reason for incomplete eradication may be that the residence time of antimicrobial agents in the stomach is so short that effective antimicrobial concentrations cannot be achieved in the gastric mucous layer or epithelial cell surfaces where H. pylori exist. Therefore, eradication of H. pylori may be better achieved by a therapy that improves antimicrobial agent delivery for topical activity and absorption for systemic activity. However, no in vivo eradication trials with dosage forms that prolong the gastric residence times for topical activity and have the high absorption rate in the gastro-intestinal tract have been reported. The best results so far have been achieved with the combination of a non-absorbed agent with topical activity, colloidal bismuth compounds, and a well-absorbed agent with systemic activity, amoxicillin (Van Caekemberghe and Breyssens, 1987, Antimicrobial Agent and Chemotherapy, pp 1429–1430). But clearly, therapy for H. pylori infections is still suboptimal.
In addition to a bactericidal effect, some bismuth compounds have profound effects on some of the pathogenic mechanisms whereby H. pylori damage the mucosa. Bismuth compounds are potent and non-specific enzyme inhibitors. In vitro studies have suggested that these compounds may inhibit bacterial enzymes, including lipases, proteases, and glycosidases synthesized by H. pylori. Inhibition of bacteria enzymes and maintenance of an intact viscoelastic gel coating is thought to be related to the therapeutic action of bismuth compounds for H. pylori associated gastritis and peptic ulcers.
Bismuth compounds block the adhesion of H. pylori to epithelial cells. Shortly after oral administration of these compounds, the organisms were located inside, rather than underneath, the mucus gel. This was thought to result from loss of adherence to the apical membrane of the gastric epithelial cells. Bismuth, in the form of electron dense bodies, was seen to be deposited on the surface and within the bacterial cell. But unfortunately, intramucus bismuth concentrations often fall below the mean inhibitory concentration of bismuth compounds for H. pylori, because of the diluting effects of food, long disintegration time of commercial tablets at pH<1.1, and bismuth precipitation due to insolubility in acidic environment. Additionally, H. pylori inactivated by exposure to growth-inhibiting concentrations of bismuth compounds can remain viable for several hours and, therefore is capable of resuming normal growth when bismuth is removed.
The inhibitory factor(s) in bile was markedly reduced by acidification followed by centrifugation to remove precipitated glycine-conjugated bile acids and was completely eliminated by use of the bile acid-sequestering agent cholestyramine. These results are consistent with the notion that acidic conditions in the duodenal bulb would serve to precipitate inhibitory bile acids (or other inhibitory substances) and allow H. pylori to grow in an otherwise hostile environment. The observations related to reflux gastritis by D. Y. Graham (Osato et al., 1999, Digestive Diseases and Sciences 44(3):462–464) can be extended to possibly understand the relation between acid secretion and duodenal ulcer. Low pH in the duodenal bulb in patients with duodenal ulcer disease associated with high acid secretion, rapid gastric emptying, and local production of acid would both promote development of gastric metaplasia and precipitate the deleterious glycine-conjugated bile acids, allowing H. pylori to colonize and thrive.